The present invention relates to a process for immobilizing microorganisms or enzymes, and more particularly to an immobilization process for preparing microbial or enzymatic beads by using polyvinyl alcohol (PVA).
In the years since 1980, the immobilization methods of microorganisms or enzymes, in which various natural or synthetic polymeric substances are used to bring about the immobilization of the activated microorganisms or enzymes, have received a great deal of attention and achieved some success in the industrial application, as exemplified by their applications in the production of biochemical products such as high fructose syrup, 6-APA, L-amino acid, etc. The representative polymeric substances that are commonly used in preparing the immobilized microbial or enzymatic beads include such substances as polyacrylamide, K-carrageenan, sodium alginate, agar, etc. Polyacrylamide is commonly used in view of the fact that it is a rather inexpensive material; nevertheless it is not suitable for use in immobilizing the living cells of microorganisms on the grounds that its monomer is toxic and that it is difficult to form beads of spherical shape, which is a desirable form characteristic for a continuous reactor. Therefore, polyacrylamide may be replaced by K-carrageenan, which is less toxic and is relatively easy to form spherical beads. However, K-carrageenan is an expensive commodity. Sodium alginate is an inexpensive material, which forms spherical beads easily. But it has a shortcoming that its gel strength is rather instable in a reaction solution containing phosphate, and sodium or potassium cations. The agar is a gelatinous material having a lack of sufficient mechanical strength to sustain a prolonged operation. The immobilization techniques of microorganisms afford a great deal of potential of improving the production process of biochemical products and the wastewater treatment. It is, therefore, important that we are in need of developing a new and inexpensive immobilization material which is not toxic to microorganisms and has an strong gel strength so as to ensure that the bead formation is successful.
Polyvinyl alcohol (PVA) is a water-soluble polymer; it has advantages that it is nontoxic to both human being and microorganisms, and that it is provided with a mechanical strength sufficiently strong enough to ensure the success of bead formation, and further that it is a polymeric substance which is commonly used in industry and produced in quantity economically. Therefore, PVA is an ideal substance for use in immobilizing microorganisms.
Various patented methods of immobilizing microorganisms by PVA have been disclosed in recent years, as exemplified by the Japanese patent applications Kokai 57-14129 (1982) and 61-139385 (1986) in which the entrapment methods are characterized in that the gelations of the mixture containing the PVA aqueous solution and the microorganisms are carried out by the methods associated with the freezing and thawing technique. Another Japanese patent application Kokai 1-454372 (1989) discloses a method, in which the mixture of the PVA aqueous solution and the microorganisms is exposed to the ultraviolet radiation so as to form a photocrosslinking gelation. Another gelation technique has to do with the formation of crosslinking structure by permitting the mixture of the PVA aqueous solution and the microorganisms to make contact with and the microorganisms to make contact with a saturated boric acid solution, as disclosed in an article by Susumu Hashimoto and Kenji Furukawa, entitled "Immobilization of Activated Sludge by PVA-Boric Acid Method", Biotechnology and Bioengineering, Vol. XXX, pp 52-59 (1987). In spite of the fact that the above-mentioned methods of the prior art can be used to prepare the immobilized beads having a relatively good gel strength, they have shortcomings that call for further improvement. The freezing and thawing method mentioned above is defective in that it is a tedious and costly process requiring the materials to be frozen, defrosted and dehydrated and that it must be carried out at the temperatures in a range of -30.degree. C. to -80.degree. C. The photocrosslinking method is not suitable for use in immobilizing microorganisms in view of the fact that it is generally used in making thin membranes. The PVA-boric acid method requires that the mixture of the PVA solution and the microorganisms remains in contact with the boric acid aqueous solution for a period of 12-24 hours so as to form beads having a relatively strong gel strength.
In short, the prior art described above share the following shortcomings. In the first place, they are time-consuming and complicated, thereby resulting in a substantial increase in the expenditure for largescale production facilities without increasing the productivity. Secondly, an environment in which cryogenic temperature, vacuum and boric acid are present is antagonistic to the living microorganisms intended to be immobilized. The Japanese patent application Kokai 64-5490 and 64-5491 (1989) disclose the methods in which the sulfate aqueous solution is used in place of the boric acid aqueous solution. Such methods are effective in shortening the time that is required for the immobilization process to be brought to a conclusion. However, such methods use the gel solutions having a relatively high concentration. Therefore, such methods must be carried out in the presence of an aqueous solution containing 30% of sodium sulfate or 70% of ammonium sulfate. If the concentration is too low, the bead formation and the gel strength will not be satisfactory. Therefore, such methods are costly. In addition, a high salt concentration used in the gelation process has an adverse effect on metabolism of microorganisms intended to be immobilized.